The family EnterobacteriaCeae include bacteria that are characterized as small, gram-negative, oxidase negative, non-sporulating rods that are capable of fermenting glucose. These microorganisms are found in habitats such as the intestinal tract, soil, water, vegetables and other food sources such as meats and dairy products. Some members of this family are known human pathogens such as bacteria of the genus escherichia, salmonella, shigella and enterobacter.
Classical methods for determining the presence and number of Enterobacteriaceae in a sample are time consuming, tedious and labor intensive. Typically, a technician must prepare reagents and nutrients, mix the nutrients with agar, heat the mixture, pour the mixture into a petri dish, obtain a test sample, dilute the test sample, add an aliquot of the diluted sample to the agar, allow the agar to gel, incubate the inoculated plate for 24-48 hours and finally count the number of growing bacterial colonies in the petri dish. If needed, confirmatory tests may also need to be performed in order to particularly identify specific bacteria. Products and processes which reduce preparation time and allow reliable detection and colony count of Enterobactefi.aceae in a sample would clearly be welcomed by those working in this field.
One example of a product which greatly simplifies the above preparation time is thin film, dry culture medium devices for growing microorganisms that are described in U.S. Pat. No. 4,565,783 to Hansen et at., U.S. Pat. No. 5,089,413 to Nelson et at. and U.S. Pat. No. 5,232,838 to Nelson et al. In a representative thin film device, a cold-water soluble dry powder containing a gelling agent, microbial growth nutrients and minerals and an indicator dye is coated on a waterproof substrate. The waterproof substrate is covered with a foam spacer which provides an inoculation area. A transparent, read-through cover sheet that is coated on an inner surface with an acrylate adhesive containing an indicating dye and powdered gelling agent is attached to the foam spacer.
When the device is used, a predetermined amount of an aqueous sample is typically placed in contact with the coated substrate in an area defined by the foam spacer and the cover sheet is placed over the sample and substrate. The aqueous sample hydrates the soluble dry powder which then forms a gelled medium capable of sustaining microbial growth. During the growth period, the indicator dye adhered to the cover sheet reacts in the presence of viable microorganisms to give a detectable response that allows visualization of bacterial colonies which are grown on the culture device. Different types of thin film dry culture medium devices are commercially available as PETRIFILM thin film, dry culture medium plates from 3M, St. Paul, Minn.
The thin film, dry culture medium devices are much simpler to use than conventional gelled agar mediumpetri/dish systems because there is no need for the user to heat and mix the growth medium, agar and other reagents and then add the mixture to petri dishes or pour plates. In addition, the devices of Hansen et al. are compact and easily disposed of and therefore are easier and safer to use.
For example, a culture medium which may be used in a thin film plate in order to provide a rapid count of coliform bacteria is reported in U.S. application Ser. No. 08/062,311, filed May 5, 1993, now U.S. Pat. No. 5,364,766 to Mach et al. In this document, an aliquot of the sample containing coliform bacteria is added to a culture medium comprising tryptose, lactose, sodium chloride, bile salts, and an excess amount of a pH indicator, phenol red, which provides a high concentration of phenol red in close proximity to the bacteria growing in the medium. The use of the reported medium and high concentration of phenol red allow the detection and count of coliform bacteria in less than 24 hours. The reported medium, however, is hampered by diffusion of the pH indicator through the medium as the device is incubated. More specifically, the presence of coliform bacteria is initially detected by a visual color change of the phenol red indicator from a red to a yellow color in a zone around the growing microbial colony that is caused by the production of organic acids by the growing microorganisms. As the growing bacteria continue to produce organic acids that generate the colored zones, the colored zones increase in size and begin to merge with the colored zones of surrounding nearby colonies. When enough growing, acidproducing colonies are present, the medium may eventually completely change color from red to yellow. When the color of the medium completely changes from red to yellow after about 24 hours, it is possible detect a second color change using another indicator in the medium.